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Meeting Report
Organ-on-Chip in Development:
Towards a Roadmap for Organs-on-Chip
doi:10.14573/altex.1908271
1 Introduction other hazard risks of various substances under increasingly strin-
gent regulatory requirements (Marx et al., 2016).
Organ-on-Chip is considered a potentially game-changing tech- Organ-on-Chip (OoC) is increasingly regarded as a potential-
nology born from the convergence of tissue engineering and mi- ly game-changing technology for these problems (Bahinski et al.,
crofluidic technology. Organ-on-Chip devices (OoCs) are ex- 2015) and able to meet the needs of different stakeholders (Mid-
pected to offer effective solutions to persisting problems in drug delkamp et al., 2016). In spite of its promise (Zhang and Radis-
development and personalized disease treatments. This report sur- ic, 2017), pharma has nevertheless remained cautious to invest
veys the current landscape in research, development, application, in this new technology, presently awaiting evidence of its add-
and market opportunities for OoCs towards establishing a glob- ed cost-benefit value and whether it could represent a feasible
al and multi-stakeholder OoC ecosystem. Based on panel discus- route to precision medicine and improved patient stratification. It
sions held at the Vision Workshop (Stuttgart, 23 May 2018) orga- is thus necessary to bridge the gap between the potential of OoC
nized by the EU ORCHID consortium, as well as on additional systems and their worldwide acceptance. Defining the putative
bibliometric study, market analysis, and expert interviews con- benefits of OoCs and how these can be proven and achieved is the
ducted within the EU ORCHID project, we outline perceived un- preamble for an OoC roadmap – which is one of the aims of the
met needs, key challenges, barriers, and perspectives of the field. ORCHID project.
We finally propose recommendations towards the definition of a The Horizon 2020 FET-Open project Organ-on-Chip In De-
comprehensive roadmap that could render OoCs realistic models velopment (ORCHID) started in 2017 with the goal of creating
of human (patho)physiology in the near future. a roadmap for OoC technology and of building a network of ac-
ademic, research, industrial, and regulatory institutions to move
1.1 The healthcare challenge and the OoC roadmap OoCs from laboratories into general use to benefit the citizens
A critical problem in the development of effective disease treat- of Europe and beyond. The ORCHID Consortium is a collabo-
ments is the paucity of adequate model systems to identify drug ration between 7 partner organizations from 6 European coun-
targets, screen toxicity, predict clinical drug efficacy, and ascertain tries – namely: Leiden University Medical Center (coordina-
the effects of active substances in humans (Marx et al., 2016). Tra- tor, The Netherlands), Institute for Human Organ and Disease
ditional animal models (McGonigle and Ruggeri, 2014; Smirno- Model Technologies (hDMT, The Netherlands), Delft Univer-
va et al., 2018) or conventional cell cultures (Pampalonie et al., sity of Technology (TU Delft, The Netherlands), Commissar-
2007; Watson et al., 2017) often do not accurately mimic human iat à l’Energie Atomique et aux Energies Alternatives (CEA,
physiology and thus tend to poorly recapitulate human disease France), imec (Belgium), Fraunhofer Institute for Interfacial
pathophysiology or accurately predict in vivo responses to med- Engineering and Biotechnology (Fraunhofer IGB, Germany),
ical treatments. This is a major cause of late drug failures in clin- and University of Zaragoza (Spain) – and engages an interna-
ical trials, high costs of new drugs, and lack of medication for tional advisory board of world-renowned experts from the OoC
some diseases (Marx et al., 2016). In addition, translational issues field1. As part of the project, these and other experts were asked
(McGonigle and Ruggeri, 2014; Smirnova et al., 2018) and ethi- to share their views on the state-of-the-art, unmet needs, chal-
cal questions raised by animal use increase the pressure to mini- lenges, and barriers of the field. The results of the expert inter-
mize animal experimentation. For these reasons, the pharmaceu- views (refer to Section A.2 of Appendix2 for the full list of in-
tical industry is looking for new ways to improve the drug devel- terviewees and experts that contributed to ORCHID), togeth-
opment process (Esch et al., 2015), assess drug toxicity (Ewart et er with the results of bibliographical, bibliometric, and market
al., 2018), and identify effective and personalized treatments; bio- analyses of OoC technology (see the Appendix2 for a descrip-
medical researchers require better systems to model diseases to tion of the analytical methodology), were combined with the in-
improve understanding of their mechanism and etiology; and cos- sights and conclusions of the ORCHID Vision Workshop, held
metics, chemical, food and other industries are in need of physi- in Stuttgart on May 23, 2018. The goal of the workshop was to
ologically relevant human models to determine toxicological and define the pillars of a European OoC roadmap, including the
1 https://www.h2020-orchid.eu
2 doi:10.14573/altex.1908271s
650 ALTEX 36(4), 2019
Meeting Reports
Fig. 1: Simplified synopsis of the OoC value chain, according to the ORCHID analyses
definition of specific goals and their feasibility for concrete de- ervoir. In either case, the coupling mimics in vivo physiological
ployment of the OoC technology. coupling and provides appropriate cell-to-fluid volume ratios and
This paper outlines the results of the Vision workshop and of flow distributions to create realistic in vitro models of subsystems
ORCHID’s updated analyses, discussions among experts, con- of the human body (Ronaldson-Bouchard and Vunjak-Novakov-
clusions and recommendations, with a view to creating a road- ic, 2018). Human(-body)-on-Chip (HoC) systems go beyond this
map fostering a thriving global OoC ecosystem. An early draft of and aim to emulate whole organismal physiology by integrating
this document3 was released at the International OoC Symposium many relevant single-organ models (Marx et al., 2016).
(IOOCS, Eindhoven, the Netherlands, 8-9 November 2018). The As implied by these definitions, OoC technology is poised on
present version features extended content and reference list, and converging advances in tissue engineering, semiconductor and
discloses the full list of contributing experts (see Appendix2). polymer microfabrication, and human cell sourcing. The associ-
ated value chain emerging from the ORCHID analyses (Fig. 1)
1.2 Organ-on-Chip: Definition, key features, highlights the need for multidisciplinary approaches to imple-
and value chain ment OoCs and for facilitating dialogue between academic and
According to the interviewed experts, and as ratified during the industrial developers as well as stakeholders such as clinicians,
ORCHID workshop, an OoC can be defined as “a fit-for-purpose patients, regulators and different end-users. All these disciplines
microfluidic device, containing living engineered organ substruc- are represented in the ORCHID Consortium.
tures in a controlled microenvironment, that recapitulates one or
more aspects of the organ’s dynamics, functionality and (patho)
physiological response in vivo under real-time monitoring”. 2 State-of-the-art
OoCs can be classified into two types with complementary
goals and distinct complexity: (i) single-organ systems, emulat- 2.1 An internationally expanding field of research
ing key functions of single tissues or organs, and (ii) multi-organ The OoC field emerged from the convergence of microflu-
platforms, combining multiple OoCs to reproduce the systemic idics and tissue engineering research. Whereas in the ear-
interaction and response of several organ models within a single ly 2000’s, patents and publications in the field mainly focused
system. Multi-OoCs link individual OoCs through tubing or mi- on microfluidics and associated fabrication techniques, more
crofluidic channels (Vernetti et al., 2017), often coated with en- recently advances in stem cell biology, combined with de-
dothelial cells or with functional coupling provided by transfer of cades of fundamental biological studies in cell signaling and
effluent from one organ’s effluent reservoir to another’s input res- biomechanics, have accelerated the development of OoCs.
3 http://www.hdmt.technology/download/?id=9618 (accessed 18.09.2019)
ALTEX 36(4), 2019 651
Meeting Reports The ORCHID bibliometric approach reflected this increas- clinicians, and end users, from both academia and industry. ing activity by finding evidence for a rise in both dedicat- Originating in the USA, which pioneered the development of ed patents and publications with a compound annual growth customized cellular microenvironments to capture the structur- rate (CAGR) of +46% over the last ten years (123 patents al complexity of organs, this worldwide interest is increasingly and publications in 2007, compared to at least 390 in 2017) engaging Europe, which has a strong track record in tissue engi- (Fig. 2a). Such continuous growth, driven by the huge market neering and microfluidics, and for which OoCs could represent potential, was made possible by the concomitant diffusion of a new growth opportunity (Fig. 2b,c). The Asia-Pacific region multidisciplinary approaches supported by improved dialogue (APAC) also contributes substantially to the field with both es- between developers such as biologists (cell culture, physiology), tablished contributors and newcomers, supported by exponen- engineers (microfluidics, biosensing systems), material scientists tial growth in technological and biological research, especially (microenvironment, substrates), but also regulators, patients, in Korea, China and Japan. Fig. 2: The ORCHID bibliographic analysis a) Temporal evolution of the number of OoC-related patents and scientific publications over the 2000-2017 period. The imported database includes 3586 documents, mostly publications (83% versus 17% of patents). The 592 identified patents include granted (38.9%), pending (25.8%) and fallen or revoked (35.3%) patents. Year 2015: latest complete year for patents (18 months are needed to publish the patent application). Year 2016: latest complete year for publications (documents were imported in November 2017). CAGR, Compound Annual Growth Rate. b) Temporal evolution of the number of publications per region. c) Temporal evolution of the number of patents per region. 652 ALTEX 36(4), 2019
Meeting Reports Fig. 3: Comparison of key specifications and features across available biological models Animal models can include human cells – consider for instance patient-derived xenografts (PDXs) in nude mice, and nude mice with humanized immune systems. Biosensors include TEER, genetic reporter cells, cell impedance spectroscopy, and microphysiometry. Orange boxes, not yet demonstrated; light blue boxes, possible or envisioned in the near future; blue boxes, in use. 2.2 OoCs hold promise as avatars for native gration of biosensors for real-time data collection, and possibility functional tissues of interfacing with both targeted (Vernetti et al., 2017) and untar- Dynamic R&D activities have been translated into technological geted mass spectrometry (Brown et al., 2016) provide OoCs with advances in both microfluidics and tissue engineering, provid- important advantages over other models (Marx et al., 2016; Wik- ing OoCs with key added values towards more accurate views of swo et al., 2013a). The tightly monitored regulation of the cellular what happens in the human body compared to other models (Fig. environment and homeostasis should facilitate long-term cell cul- 3). OoC technology allows reconstitution of the microarchitec- ture, possibly over periods of months. In addition, the great diver- ture of the organ supported by the design of a dedicated mechan- sity and range of complexity achievable in OoCs offer the oppor- ical context matching the shape, surface pattern and stiffness of tunity to optimize or even customize the design for targeted stud- organ-specific microenvironments. Precise control over microflu- ies, paving the way to personalized medicine. idic flow rate enables optimal oxygenation and nutrition supply, In light of these potential benefits, the main applications of and affords not only long-term viability of healthy tissues but al- OoCs are seen to range from toxicity testing to human disease so an efficient circulation of immune cells, antibodies, biochemi- model building and drug discovery. Users are increasingly from cal signaling molecules and metabolites, and the ability to collect a wider spectrum of fields and include not only biomedical re- small volumes of fluid containing secreted cell products for analy- searchers attempting to understand disease mechanisms and eti- ses. Continuous perfusion and mechanical stress help to build dy- ology but also industrials with different profiles – pharmaceutics, namic tissue models, which are physiologically more relevant than biotechnology, cosmetics, chemistry and environment – all inter- conventional static cell cultures and enable spatiotemporal control ested in predicting efficacy and safety of compounds for humans. of chemical gradients and mechanical cues to study the influence Some researchers are even further, already exploiting aspects of of the microenvironment on the cells. Moreover, OoCs allow pre- the long-term potential of OoCs for studying developmental bi- cise investigation of specific tissue-tissue interfaces, organ-organ ology principles and optimizing differentiation of iPSCs into var- communication and biological events that cannot be monitored in ious terminally differentiated cell types in situ for application to animals or human patients. Their small size, ability to control the and understanding of regenerative medicine and tissue replace- microenvironment, optical access at high spatial resolution, inte- ment purposes. Existing models and proposed applications have ALTEX 36(4), 2019 653
Meeting Reports
been comprehensively reviewed previously (Marx et al., 2016; ademic teams are concentrated within pioneering American hubs
Zhang and Radisic, 2017; Ronaldson-Bouchard and Vunjak-No- such as the ecosystems of Boston (Wyss Institute, MIT, Harvard,
vakovic, 2018; Zhang et al., 2018). Brigham and Women’s Hospital) and California (Berkeley, UC-
Nevertheless, OoCs remain in need of substantial scientific ev- LA, UCI, Stanford), and the universities of New York (Cornell,
idence of correlation of readouts with clinical and human physi- Columbia) and Pennsylvania (Drexel, Pittsburgh). These hubs
ological behavior. In particular, the metrics to be considered and have tended to work especially on brain, lung and heart to ad-
their corresponding readouts largely remain to be defined. They dress toxicological issues, but also on muscle, vasculature and
are a prerequisite for demonstrating the relevance of OoCs com- bone marrow. The APAC region has historically included play-
pared to other traditional approaches (e.g., single cell type, single ers like the University of Seoul (Korea), the Chinese Academy
monolayer; see also Section 3.1) and to develop a roadmap of of Science (Beijing, China) and the University of Tokyo (Japan),
how they could be implemented in decision-making processes. leading chemical, physical and biological engineering approaches
Encouraging instances in the recent literature include verification to emulate in particular brain, liver and lung tissues. The attractive
of detectable penetration of trimethylamine-N-oxide in human ce- market has also driven the repositioning of key European players
rebrospinal fluid by Del Rio et al. (2017), first predicted by Ver- in microfluidics and cell culture, such as the Fraunhofer Institute
netti et al. (2017) using a multi-OoC system, and the joint work (Germany), the University of Twente (The Netherlands), the CEA
of the Wyss Institute and Janssen Pharmaceuticals on modelling (France), the Technological University of Compiègne (part of the
the toxicity of a monoclonal antibody therapeutic (Barrile et al., CNRS, France), Jena University (Germany), and the Swiss Feder-
2018) (see Section 4.1). al Institute of Technology (EPFL, Switzerland), which largely fo-
cus their research on the modelling of brain, liver, kidney and skin
2.3 Rapid development of OoCs driven by for toxicological purposes.
academics supplying multifaceted industrials These academic ecosystems supply industrials from various do-
The OoC market gathers many of its participants from different mains, which may coexist to play a dedicated role in the OoC val-
disciplines, and continuously evolves due to rapid technological ue chain (Fig. 5, 6): i) pharmaceutical companies; ii) companies
advances and the field’s strong interdisciplinary requirements. specialized in microfabrication, imaging and electronics/robotics;
The ORCHID bibliometric analysis identified at least 650 play- iii) lab-on-chip manufacturing companies; and iv) OoC companies
ers (the top 30 are shown in Fig. 4). Many of the most active ac- (i.e., commercializing fully operational OoC). Indeed, the devel-
Fig. 4: The top 30 academic players in the OoC field according to the ORCHID bibliographic analysis
These, out of 650 identified academic players, published more than 34 documents over the 2000-2017 period. Blue bars, number of
patents and publications. The size of the circles is directly proportional to the number of identified documents published each year.
654 ALTEX 36(4), 2019
Meeting Reports Fig. 5: Overview of the top 30 companies active in the OoC field Companies are characterized as pharmaceutical (a), lab-on-chip (b), microfabrication, imaging and electronics/robotics (c), OoC companies proper (d) and others (e) – which published more than 2 documents over the 2000-2017 period, according to the ORCHID bibliometric study. Note: Charles Draper Lab is a non-profit and non-stockholding organization with a strong R&D activity. For this reason, it is not listed among companies. opment of OoCs has been supported over the past several years itized single-tissue development. Very few of them are already in by pharmaceutical companies (e.g., GSK, Roche, AstraZeneca) the production and commercialization phase, the others being pre- searching for alternative or better predictive models, especially dominantly engaged in an iterative process with end users to test for the lung, the liver, and the digestive and nervous systems. OoC different OoC solutions, i.e., manufacturing prototypes and pro- manufacturing and peripheral instrumentation require the concom- ducing small series in-house. For instance, Emulate formed vari- itant involvement of supply companies specialized in microfabri- ous strategic partnerships with pharmaceutical industries (Roche, cation, imaging, electronics and robotics (e.g., Seiko, Philips, Carl Takeda Pharmaceuticals, Merck, Janssen) to improve its solu- Zeiss; Fig. 6), in particular for OoC characterization and monitor- tions, but also with the Food and Drug Administration (FDA) to ing. Traditional lab-on-chip manufacturing companies (e.g., Aline, evaluate and qualify the use of its technology for toxicology test- Micronit, Microfluidic Chipshop, Minifab) consider OoCs as a ing. OoC companies need both external partners and high finan- promising growth opportunity. They are starting to partner with cial investments to go through the prototyping phase and to scale OoC companies and propose tailored approaches to support hard- up the production. Impressive growth is also made possible by a ware development to scale up and standardize chip production. switch towards the small- or medium-scale production volume, OoC companies are mostly start-ups founded by ex-academic as shown by Mimetas, which is mass-producing its OrganoPlate® teams (e.g., CN Bio, TissUse, Emulate, Mimetas; Fig. 6) (Zhang after only 4 years of existence, and is supplying nearly all major and Radisic, 2017). According to the Yole Développement mar- pharmaceutical companies. ket report (Yole (market) report in the following), only 18 private The current market positioning of OoC companies is tightly as- OoC companies were in the market in 2017 (Roussel et al., 2018). sociated with the type of devices developed and their “technolo- Among the most active, TissUse and Nortis are characterized by a gy readiness level”. Three different business models are present- multi-tissue R&D activity, whereas the others have largely prior- ly distinguishable: i) ready-for-culture microfluidic devices (chips ALTEX 36(4), 2019 655
Meeting Reports Fig. 6: OoC players’ positioning along the value chain (see Fig. 1) The Figures represent the number of academic players (blue circle) and OoC companies (purple circle) identified via the bibliometric approach. Academics and OoC companies show a dedicated expertise for the development of fully operational OoCs, whereas the other industrials are supporting their development through technological expertise, financing and/or partnerships. For each profile, only the top players are shown. (Note: Charles Draper Lab is a non-profit and non-stockholding organization with a strong R&D and training/educational activity. Therefore, here it is not considered as a company). and/or plates); ii) fully operational OoCs, including the microflu- commercialization and market penetration. They need to entertain idic device and cell culture integration; and/or iii) a full-service tight relationships with academia to ensure a continuous techno- offering to perform in-house tests requested by end users. Com- logical sourcing and to remain competitive. panies may switch between these business models; and according to the Yole market report, the large majority of OoC companies 2.4 Public and private investments are required are not decided whether they should sell the devices or offer test- to move OoCs from bench to bedside ing services (Roussel et al., 2018). Starting with a service-based Both public and private sources have substantially funded OoC business model may help to build strong relationships with cus- start-ups and research groups during the last few years. For in- tomers, whereas getting direct input and better understanding of stance, in 2012 the US National Center for Advancing Transla- customers’ needs enables the joint development of effective OoC tional Sciences (NCATS), in conjunction with the National In- platforms. From an end-user point of view, the service-based of- stitutes of Health (NIH) Common Fund, invested $70M over a fer could be of great value for early stage screening, when there 5-year period to launch the Microphysiological Systems or Or- is a large number of different compounds to be tested, especially gans-on-Chips Program (Low and Tagle, 2017). The aim of this in terms of logistic organization. It is mostly more convenient to program – part of a coordinated effort between the NIH, the ship a drug or a chemical compound than a cell culture. To under- Defense Advanced Research Projects Agency (DARPA) and the stand a complex biological interaction, though, some customers, FDA – was to accelerate the development of human OoCs that particularly pharmaceutical companies, may be more interested in “will improve the reliability to identify human drug toxicities and buying the OoC to do their experiments in-house. predict the potential efficacy of a drug in a human population Whatever the business model of choice, the OoC companies prior to use of the drug in late-stage clinical studies” (NCATS know that minimizing the operational complexity is crucial for website). The program awarded funds to three main multi-partner 656 ALTEX 36(4), 2019
Meeting Reports
Fig. 7: Market forecasts for OoC
devices over the 2017-2022 period
proposed by the Yole Développement
market report
The optimistic (a) and realistic (b) OoC
scenarios show that the market could
grow at an expected CAGR of 57% to
38% to reach respectively $117M to
$60M in 2022. The estimated revenues
are split between direct devices sales
(ready-for-culture devices and fully
operational OoCs, orange bars) and full-
service sales (tests performed in-house,
yellow bars). CAGR, Compound Annual
Growth Rate. (Reproduced with permission
from Roussel et al., 2018).
initiatives, led respectively by the Wyss Institute and MIT. Sim- total of $27.65M (3 rounds in 2014, 2015 and 2018), and TissUse
ilarly, the Space and Naval Warfare Systems Center, Pacific (Germany) with $4.6M in 2015. The Netherlands funded a national
(SSC Pacific), on behalf of the Defense Threat Reduction Agen- OoC initiative (called NOCI) in 2016 with a budget of €18.8M for
cy (DTRA), awarded a $24M federally funded grant in 2013 to 10 years; a H2020 Innovative Training Network project on OoC
a project led by Wake Forest Baptist Medical Center’s Institute (EUROoC), coordinated by Fraunhofer IGB, was recently grant-
for Regenerative Medicine to develop a body-on-a-chip mod- ed €3.94M for 4 years. Globally, US companies are far ahead in
el that could determine responses to harmful agents and aid the fundraising: the US government is particularly active in support-
development of potential therapies (DTRA eX Vivo Capabilities ing the OoC field, especially through DARPA, NIH, DTRA and the
for Evaluation and Licensure “X.C.E.L.” program). In 2014 the Environmental and Protection Agency (EPA), and most recently
DTRA additionally funded the $19M multi-institutional project the National Aeronautics and Space Administration (NASA) and
Advanced Tissue-engineered Human Ectypal Network Analyzer the Center for the Advancement of Science in Space (CASIS), with
(ATHENA) to develop a system interconnecting four organ con- investments of more than $224M over the past 5 years.
structs, namely for liver, heart, lung and kidney.
In the meantime, there is growing confidence among private in- 2.5 Market forecasts: From an emerging market
vestors in OoC technology. In particular, 2018 was prolific for fund- towards exponential growth
raising, with $36M and $20.5M secured by Emulate and Mime- The Yole market report estimates the combined sales of OoC de-
tas, respectively. According to the Yole market report, the most ef- vices and services at no more than $7.5M in 2016, with the poten-
ficient company in fundraising is Emulate with 4 rounds in 4 years tial to undergo an impressive growth and become a multi-billion
for a total of $93M (Roussel et al., 2018). Other US companies dollar market in the mid- to long-term, in view of the possibility
have also raised several millions of dollars: these include Hµrel that OoCs might help the industry to save billions of dollars every
Corporation ($9.2M in 2013), and Nortis (more than $8M in total, year, especially by bridging the translational gap between preclin-
with a first round of $2.65M in 2014). In Europe, 2 companies have ical and clinical studies required during drug development (Rous-
also succeeded in raising funds: Mimetas (The Netherlands) with a sel et al., 2018). As already indicated, only few players are cur-
ALTEX 36(4), 2019 657Meeting Reports
rently in the production and commercialization phase; and while have greater similarities to normal or diseased human tissue than
pharmaceutical and cosmetics companies are engaged in an itera- the widely used immortalized cell lines.
tive process to test different OoC solutions, they are conservative
and might need time to adopt the technology widely. This overall OoC can pave the way to personalized medicine and
context led Yole Développement to detail both a realistic and opti- de-risking drug development
mistic scenario, in which the market could grow at a CAGR from The limited ability of preclinical cell cultures and animal mod-
2017-2022 of 38% to 57% to reach $60M to $117M, respectively els to predict drug efficacy and safety reliably during the later hu-
in 2022 (Fig. 7; for further market data, see Roussel et al., 2018). man clinical trial stage (Abaci and Shuler, 2015) is not only dis-
In the optimistic scenario, based on the companies’ forecast- appointing but also ends up wasting billions of dollars each year
ed revenues (Fig. 7a), the Yole report considers that if all con- in development costs for ineffective drugs and slows down the in-
ditions are met, i.e., industrial adoption speeds up, OoC compa- troduction of new medical treatments in the clinic. Spending on
nies can overcome technical challenges and upscale production, drug development increased by orders of magnitude over the past
858k units per year could be manufactured by 2022, correspond- 20 years, but the number of drugs approved for market per year
ing to an overall market of $117M (Roussel et al., 2018). The ser- actually declined (Zhang and Radisic, 2017). Many compounds
vice-based market is presently expected to remain much smaller with high potential health benefit are eliminated early in develop-
than the device sales market, and whether it will be sustainable ment due to the poor predictability of preclinical models, i.e., they
in the long-term by OoC start-ups is unclear. However, the ser- may well be effective in humans but not in animal models as well
vice-based market could grow significantly through involvement as the converse, which is more frequent. Some drugs have actual-
of contract research organizations (CROs), which provide a mul- ly been withdrawn shortly after entering the market because their
tiplicity of services for pharmaceutical companies and other cus- toxicity (mainly on heart, liver and kidney) was not predicted.
tomers, if they begin to take over from OoC developers and offer Late-stage failures cause catastrophic losses while significantly
large-scale or high-throughput services with OoC models. driving up cumulative costs and patient risk. If OoCs could be po-
In the realistic scenario (Fig. 7b), the Yole report forecasts that sitioned as proven predictive tools for preclinical screening, they
the majority of OoC developers will face issues if the demand might represent the ideal solution for the pharmaceutical industry
grows (Roussel et al., 2018). It is noted in particular that scaling to eliminate ineffective drug candidates as early as possible and
up the production will likely slow down the growth of OoC com- curb the costs of drug development (Zhang and Radisic, 2017).
panies during the period 2018-2021. Most of them will have to As expected, the OoC applications most cited in scientific lit-
switch from PDMS prototyping and small-series production to erature and in the ORCHID expert interviews lie in the pharma-
large-scale manufacture in other materials (glass, polymer injec- ceutical drug development process (Abaci and Shuler, 2015), and
tion molding), requiring redesign steps that will be expensive for are related largely to safety assessment and efficacy testing (see
the relatively small companies involved. In this scenario, the OoC also Watson et al., 2017). The main idea shared by all players is
market will grow from $7.5M in 2016 to $59.7M in 2022 because that OoCs may enable more rapid, accurate, cost-effective, and
only few companies have already managed to scale up their pro- clinically relevant testing of drugs. According to a recent scenar-
duction. The revenue fraction deriving from services is higher io-based cost analysis conducted in the context of the ORCHID
than for the optimistic scenario because OoC companies will fo- project, experts expect a positive budget impact, reaching a re-
cus on customized services to offset losses due to the costs of pro- duction of up to 26% in the total R&D costs for drug develop-
duction upscaling. ment (Franzen et al., 2019). While all cost drivers may be impact-
ed, savings would mostly be achieved by improving the success
rates for effective disease treatment. The R&D phases in which
3 Challenges experts expect the most benefits are lead optimization and preclin-
ical phases (see also Watson et al., 2017).
3.1 Unmet needs Data from animal studies are often poorly indicative of the hu-
Evidence for added value man situation (McGonigle and Ruggeri, 2014; Mak et al., 2014),
A notable unmet need that emerged from the experts’ interviews since animal preclinical models have limitations in mimicking the
is data-supported evidence of actual advantages of the OoC tech- complex processes specifically occurring within the human body
nology compared to existing conventional models or well-estab- (Marx et al., 2016). OoC models may potentially bridge the trans-
lished approaches to tissue engineering. Particularly, while the su- lational gap from target or lead compound discovery to market in-
periority of 3D over 2D cell cultures for, e.g., phenotypic expres- troduction and actually foster the implementation of the 3Rs, i.e.,
sion, is attested in recent scientific literature (Alépée et al., 2014), Reduction, Refinement, and Replacement of animal testing. How-
the advantages deriving from the inclusion of dynamic perfusion ever, all interviewed experts agreed that OoCs (and most other in
and in situ stimulation in OoCs are only recently becoming evi- vitro systems) are currently far from replacing all animal models,
dent (Vernetti et al., 2017; Del Rio et al., 2017) and arguably need and should be viewed mostly as complementary approaches to an-
more substantial support. In this context, incorporation of organ- imal testing. Specifically, and as evidenced in the ORCHID im-
oids (or dissociated tissue cells derived from them) may have par- pact analysis (Franzen et al., 2019), high-throughput plate-based
ticular advantages, since they represent a renewable source of cell microphysiological systems may find use in target identification,
types that are normally difficult to access repeatedly and which lead selection, and lead optimization at assay scale; chip-based,
658 ALTEX 36(4), 2019Meeting Reports
low-to-medium throughput devices may find most fitting use in resent excellent tools to investigate the onset of lung diseases (Be-
pre-clinical, single-organ toxicity, or efficacy tests; and multi-or- nam et al., 2016), such as smoking-induced chronic obstructive
gan or coupled OoC systems may target the replacement of ani- pulmonary disease and its malignant transformation.
mal models for toxicity screening, testing of pro-drugs, and phase
I / phase II clinical testing in patients – which is being prefigured 3.2 The future of OoCs lies in automation, robustness
as (pre)clinical-trials-on-chip. and integration of complexity
Replacing (pre)clinical trials and advancing towards the imple- Automation and ease of use
mentation of personalized medicine are the grand unmet needs Present OoCs are typically complicated pieces of engineering,
targeted by multi-OoC and HoC systems. OoCs pave the way whereby the microfluidic chip accommodating the cell co-cul-
to personalized medicine approaches by enabling the use of pa- tures is connected to external control and supply peripherals.
tient-specific primary and stem cell sources to capture important For this reason, ease of use of the systems and increased automa-
differences arising from genetic diversity, origin, gender or age. tion in setting up cell cultures and keeping them viable through-
For instance, OoCs can enable the development of in vitro clin- out study times are commonly perceived unmet needs in labora-
ical trials for patient populations not fit for standard clinical tri- tory practice. Ideal OoCs would be simple devices displaying us-
al designs (i.e., rare and/or pediatric diseases) or to develop drug er-friendly interfaces that facilitate the work of users with even
regimens that are optimized for specific patient biology (Ronald- limited training on, e.g., cell cultures and instrumentation. These
son-Bouchard and Vunjak-Novakovic, 2018). Along this path, devices should be easily transferrable from developers to end us-
OoCs may represent models not only capable of capturing prior ers by means of standardized guidelines concerning biomarkers
organ(ismal) knowledge and recapitulating known physiological and endpoints. In addition, they may be prepopulated with frozen
responses, but also, and even more importantly, unique tools for cell cultures ready-to-use by end users upon thaw.
unprecedented investigations and new discoveries for the further
advancement of physiology and medical science. Improving single OoCs
Among the many different organs and tissue types that can be cur-
OoCs can benefit toxicology testing rently emulated (Marx et al., 2016), the outcome of the experts’
The interviewed experts share the opinion that industries devel- interviews and of the ORCHID bibliometric analysis indicated
oping cosmetic, chemical and agro-food consumer products could a strong preference for ADME (absorption, distribution, metab-
find huge potential in OoC applications with respect to toxicologi- olism, excretion) pathway processing, including metabolic (liv-
cal hazard and risk assessment of substances for the study of, e.g., er, kidney) and digestive organs (Maschmeyer et al., 2015), along
metabolism, effects of toxicants including nanomaterials, collec- with cardiac tissue, lung and central nervous system tissues cou-
tive responses and allergies (see also Zhang and Radisic, 2017). pled to a blood-brain barrier substitute (Bhatia and Ingber, 2014).
Regulatory requirements for chemicals and cosmetics do not ac- Organ-specific needs to be met included, among others: for the
cept any significant hazard potential of substances used for hu- liver, the development of models making use of hepatocytes de-
mans. Moreover, there is rising interest in alternative models that rived from human induced pluripotent stem cells (hiPSCs; see
allow skin sensitization assessment and systemic toxicity testing also next Section for optimal cell sourcing); for the heart, effi-
for regulatory use, because all animal testing for cosmetic and fra- cient maturation protocols for hiPSC-derived cardiomyocytes to
grance products marketed in Europe was banned in 2013. China is capture the expression of adult phenotypes; for skin, establishing
meanwhile changing regulations about animal testing for cosmet- methods to derive all skin cells from hiPSCs, including, e.g., der-
ics, approved the first non-animal tested cosmetics in 2016, and mal papilla cells needed for hair generation (Abaci et al., 2017).
the authorities are being lobbied to encourage reducing animal OoC devices can perfuse vessels, thereby including the vitally im-
use. The USA has included a ban on animal testing in their 2007 portant flow of a blood surrogate to feed nutrients and remove ex-
roadmap for all industries, including the pharmaceutical, chemi- creted products through vasculature, and the accompanying shear
cal and cosmetics industries (Roussel et al., 2018). The chemical stress. The experts emphasized that, while immunogenicity test-
industry is also actively working on such strategies, especially on ing of drug candidates in animals is essentially obsolete, immune
in vitro skin sensitization testing, to avoid unnecessary reassess- and endocrine systems (Cyr et al., 2017) are missing components
ment of chemicals due to the Registration, Evaluation, Authori- required to improve the physiological relevance of OoCs (van de
zation and Restrictions of Chemicals (REACH) regulation (Marx Stolpe and Kauffmann, 2015). Different academic groups are in-
et al., 2016). Nevertheless, more formal validation efforts will be deed developing bone marrow-on-chip to integrate aspects of im-
needed prior to full industrial OoC adoption in the chemical and munocompetence in OoC-based drug safety and efficacy testing
cosmetics industries. (Marx et al., 2016). Moreover, predictive models for gametogene-
Though not mentioned by any ORCHID interviewee, the to- sis and testicular toxicity, an infrequent but severe cause of arrest
bacco industry could represent another OoC targeted segment in of drug development or “black-box labelling”, and for other spe-
the near future due to the high pressure to increase the safety of cific toxicities for which no satisfactory pre-clinical model exists
its product portfolio (Marx et al., 2016). Ethical considerations are presumed to be valuable as a niche market for OoCs.
have prompted the tobacco industry to develop new approaches The ORCHID bibliometric analysis highlighted high interest in
and tools to assess smoke-related adverse effects on the human re- OoCs as cancer model systems, considered potentially helpful for
spiratory system, primarily airways and alveoli. OoCs could rep- understanding tumor progression, metastasis formation, and in-
ALTEX 36(4), 2019 659Meeting Reports
Fig. 8: Global trends in
pathophysiological models and
tissue types according to
the ORCHID bibliometric analysis
The imported database of patents and
scientific publications was segmented
with regards to emulated tissue types (a)
and pathophysiological models (b) on a
worldwide scale.
vestigation of inhibiting strategies. (Fig. 8). With respect to me- Integrating more complexity within multi-OoCs
tastasis modelling, detailed recapitulation of the vascular base- As mentioned earlier, the development of multi-organ OoCs is en-
ment membrane remodeling has not yet been reported. The ex- visioned in the perspective of recapitulating the complexity of hu-
ponential growth of publications dedicated to OoC cancer models man physiology at organism level, which is lost in moving away
may be related to the fact that OoCs are the only systems to mod- from animal models. A recent eminent example of a multi-OoC is
el tumor cell intravasation into a surrogate blood stream or im- the Evatar system, which couples Fallopian tubes, uterus, cervix
mune cell extravasation into the tumor by combining human mi- and liver made from human tissues with ovaries from mouse tissue
cro-perfused 3D tumor models with human vasculature (Marx et to recapitulate 28-day female hormonal cycles (Xiao et al., 2017).
al., 2016). Such strong interest is also supported by epidemiologi- Early claims of a forthcoming full HoC system appear to be
cal data and the high market potential for cancer treatments. presently more realistically modulated by a technical assessment
The expert interviews also indicated that, from a patient per- of the formidable difficulties facing its achievement. The latter in-
spective, stringent unmet medical needs are represented by ways clude: (1) the onset and maintenance of self-contained homeosta-
to improve the treatment of diseases – such as cancer, dementia, sis; (2) accounting for the influence of missing organs (Cyr et al.,
kidney and rare diseases – for which there is currently no treat- 2017), particularly the hormone background (e.g., gender and re-
ment, insufficient treatment (Osaki et al., 2018), or only treat- productive hormones) and immunocompetent cells; and (3) the
ments that are excessively expensive. identification of (alternative solutions to) a single perfusion me-
660 ALTEX 36(4), 2019Meeting Reports
dium shared by the multiple cell types (each with different re- ers of endothelial cells, providing a barrier to drug absorption into
quirements) within the system. Other challenges for multi-OoC the substrate only reproducible if the cell monolayer is confluent.
and HoC systems lie in scaling the relative volumes of the indi-
vidual OoCs (Wikswo, 2014; Wikso et al., 2013b), the intercon- Optimal cell sourcing and shared culture media
nection topology, and perfusion rates which should approximate Access to abundant, good quality human cell sources represents
physiological flow configurations (Ronaldson-Bouchard and an essential technical aspect of OoCs. Multiple cell sources are
Vunjak-Novakovic, 2018). Organs involved in ADME are consid- currently available, and each cell type presents specific advantag-
ered the basis of HoC models, since they are responsible for signif- es and disadvantages. The highest clinical relevance for humans
icant homeostatic effects and are foreseen in comprehensive or- is expected through use of adult primary cells obtained directly
gan models for compound testing, for instance in skin and lung from healthy donors or patients, although this is not the only op-
models to evaluate compound concentration in urine and blood. tion (Sutherland et al., 2016). However, genetic variance between
Modelling of ADME is considered central also because inappro- individuals may arise since it is rare that cells can be obtained re-
priate drug distribution is regarded as an important cause of un- peatedly from the same donor. Adult human cells cannot always
wanted drug side effects – a global leading cause of death. For be obtained in large quantities from many tissues (e.g., skin biop-
this purpose, efforts should focus on combining a set of organs sies), or may not be readily accessible through biopsies (e.g., from
that can match the critical functions needed for drug study with brain or heart). Adult stem cells can be collected and cultured in-
a functional endothelium serving as a selective barrier (Brown et definitely from almost all endodermal organs, e.g., the organs of
al., 2015, 2016) for transport of drugs and bioactive factors (Ron- the gastro-intestinal tract and the lungs; they can also be geneti-
aldson-Bouchard and Vunjak-Novakovic, 2018). cally modified and grown in culture in large quantities, but they
are not available from all organs or tissues, and they only repre-
3.3 Technical challenges sent the epithelial component (i.e., not the stroma or vasculature).
Structural materials: alternatives to PDMS? Pluripotent stem cells (PSCs), derived from reprogramming of
Polydimethylsiloxane (PDMS) has largely replaced glass and adult somatic cells (human induced PSCs, hiPSCs in short) or de-
ceramic as the most commonly used material for fabrication of rived from early embryos (human embryonic stem cells, hESCs),
OoCs. The wide adoption of this type of silicone ensues from a can give rise essentially to all cell types of the human body, and
convenient combination of advantages, namely biocompatibility in the case of hiPSCs can be obtained from any individual (Bellin
and oxygen permeation (crucial for cell viability), optical trans- et al., 2012). On the other hand, the differentiated derivatives are
parency (permitting real-time optical inspection), tunable visco- generally phenotypically immature, similar to fetal cells. It is thus
elasticity (which supports mechanical actuation), ease of avail- a remaining challenge to develop culture conditions for the matu-
ability, and straightforward processing (no strict need for specif- ration of such cells, and to use them to model diseases developing
ic cleanroom facilities) – including deposition by casting and after birth (Avior et al., 2016). Spheroids and organoids (Fatehu-
spin-coating, functionalization, metallization, and patterning by llah et al., 2016) derived from human biopsies and hPSCs are al-
soft lithography and molding (Zhang et al., 2018). The almost so regarded as sources of pre-organized, higher-order tissues suit-
unanimously remarked disadvantage of PDMS – though com- able for inclusion in OoCs (Zhang et al., 2017).
mon to most polymers, inc luding the plastics of interconnect- While the dynamic microenvironment of OoCs, which attempts
ing tubes – lies in its lipophilicity (Vernetti et al., 2017) and re- to mimic human physiology, can support cell differentiation and
sulting non-selective absorption of hydrophobic drugs, particu- maturation better than static cell culture conditions, the co-pres-
larly those with low molecular weight (van Meer et al., 2017). ence of multiple cell types in the same microphysiological device
Non-selective absorption complicates the localization of target sharing a common culture medium may cause additional prob-
compounds in the devices and, in the absence of models predict- lems. According to the ORCHID experts, the issue of the formu-
ing drug absorption rates to PDMS and bioavailability to cells in lation of a single perfusion medium may be solved by a strict bio-
the devices, can drastically affect the interpretation of analytical chemical approach, rather than from simple media mixing. Culture
results. Moreover, drug absorption directly affects the choice of media containing, e.g., high glucose concentrations are standard
how to deliver drugs to OoCs. Drug delivery in OoCs should pref- but may not be appropriate as cells mature, and many applica-
erably replicate the physiological application in humans, e.g., di- tion-specific media are proprietary so that their composition is not
rect exposure to cells, inhalation, skin absorption, oral delivery, known. Fetal calf serum, widely used until recently to supplement
or intravascular delivery by perfusion in vessels. The limitations culture medium with growth and other factors, is presently being
of PDMS may be tolerated during device prototyping and charac- largely replaced by chemically defined media but, again, formu-
terization, but may hinder future large-scale integration unless ef- lations may not suit all cell types in co-culture. Finding common
fectively modelled. Solutions to the non-selective compound ab- media is an active area of research by media supply companies and
sorption in PDMS face a number of challenges: (1) the search for others. Blood is used as medium as well despite constraints on its
or cost of application-specific formulations of PDMS or alterna- usage for safety and other reasons. Finally, whereas the choice of
tive polymers, since most are based on polycarbonate, polysty- cell types is generally dependent on the specific organs and ap-
rene, styrene ethylene butylene styrene (SEBS), polyimide, poly- plications of interest, a related challenge concerns the appropriate
urethane or hydrogels for which intellectual property covers use representation of the diversity inherent in the human population in
through patents; or (2) the coating of polymer surfaces with lay- terms of gender, ethnic origin and age.
ALTEX 36(4), 2019 661Meeting Reports Long-term viability and multimodal real-time characterization are not fully understood. Therefore, the integration of bio-compat- could revolutionize cell culture utility ible electrical, chemical or physical sensors and actuators is highly With typical sizes of around a few millimeters, OoCs afford dis- sought-after and widely investigated (Zhang et al., 2017). For in- tinct advantages in terms of non-invasive organ monitoring and stance, trans-epithelial electrical resistance (TEER) (Henry et al., tissue investigation compared to direct investigations in human in- 2017) and fluorescein isothiocyanate (FITC)-dextran permeability dividuals or patients. The small volumes of tissues and culture me- (Brown et al., 2015, 2016) are being measured as real-time read- dia confined within the closed, controlled environment of the de- outs of tissue barrier functions; and micro-impedance tomography vices limit the dilution of xenobiotics and metabolites. Small chip is used to quantify the deflection of membranes induced by breath- volumes additionally help ensure sterile culture conditions, though ing motion (Mermoud et al., 2018). they may be also responsible for reduced throughput unless part of multi-well assays for example. A related advantage concerns Qualification for contexts of use should be preferred to validation the potential extended viability of cells in OoC devices compared Some form of validation of OoC systems is critical for their broad- to standard cultures, enabled by continuous microfluidic nutrient er acceptance and uptake in industrial and clinical settings, particu- feeding and removal of waste products. Recirculation without a larly in relation to supporting decision-making in a regulatory con- liver or kidney however may entail either use of large media vol- text. In Guidance Document 34, the OECD defines “validation” as umes or media replacement. Required model viability is consid- “the process by which the reliability and relevance of a particular ered by the experts interviewed to be sensibly dependent on the approach, method, process or assessment is established for a de- application and specific questions at hand: it can range from a few fined purpose”. Although this definition is in principle applicable hours to a few days (e.g., 3 to 8 days) for repeated dose/exposure to OoC devices used for a particular purpose, the context of OECD and acute toxicity testing; up to a few weeks for safety and effica- guidance typically relates to the validation of methods intended cy testing (e.g., 28 days for systemic testing, in accordance with to form the basis of internationally recognized test guidelines that the Organisation for Economic Co-operation and Development can be used in any of the 36 OECD member countries, implement- (OECD) guidelines for animal tests) (Maschmeyer et al., 2015); ed within a Good Laboratory Practice (GLP) quality system (as and up to several months for chronic toxicity testing or modelling/ exemplified in the OECD’s Guidance Document on Good In Vi- monitoring disease course progression in vitro, in analogy with tro Method Practices (GIVIMP)), and satisfying the conditions phase II clinical trials. Tissue viability has been hypothesized as of Mutual Acceptance of Data (MAD) between jurisdictions and being indefinitely extendable in the presence of self-contained ho- regulatory agencies. Therefore, the validation processes foreseen meostasis, particularly when sustained by vascularized intercon- in this context may be more appropriate for highly standardized nections among several organ modules. and widely applicable methods, and may probably not be suitable Online characterization of organ- or tissue-specific phenotypes, for emerging OoC devices in the near future. Moreover, validation readouts and endpoints in OoCs may include vascular contraction, per se is not considered by some experts an appropriate nor mean- cell migration, responses of genetic reporters, and metabolic path- ingful concept in this context, because it implies the existence of ways. The methodology is largely dominated by optical and flu- an accepted standard to be used to confirm or measure validity. In orescence microscopy, which are expected to remain prevalent fact, neither animal models nor human clinical effects can be uni- for the foreseeable future. A challenge for assay imaging systems vocally used as golden standard for validity. Besides, a universal is translation into high-throughput industrial settings. Incubators approach to validation is probably unrealistic, and a harmonization at body temperature are commonly used to host OoCs and reg- across contexts and cultures is expected to take too long and might ulate cell culture parameters, such as pH and CO2 levels. Com- not be possible at all. position of perfusion media, fluid flow rates, and gas pressure for Considering that the application of OoC devices in the short/me- pneumatic actuation of flexible membranes are mostly controlled dium term is most likely to be in the pharmaceutical sector, and by external, partly automated equipment. Connection of fluidic pe- will primarily target specific and well-defined contexts of use, fo- ripherals to chip or plate devices typically involves silicone, Ty- cus should be on the qualification of devices, rather than on valida- gon, PEEK or other flexible tubes, which may cause bubble for- tion in the broader sense. “Context of use” refers here to a clearly mation and non-selective drug absorption. Proper utilization of articulated description delineating the manner and purpose of use devices and peripherals typically requires specific training of us- of a tool, while “qualification” is understood as arriving at a con- ers and is expected to be regulated by standardized guidelines. In- clusion that the results of an assessment using a model or assay cluding such peripherals as integral components of self-contained can be relied on to have a specific interpretation and application in OoC devices may also raise issues. According to the experts con- product development and regulatory decision-making. Aiming for sulted by ORCHID, OoCs should be consumables whereby re- qualification of OoC devices was in fact the approach proposed in al-time sensor readout and system control are simultaneous and a recent series of workshops devoted to this important topic – held aligned. The experts recognized that real-time monitoring of bio- both in the US by the FDA and in Europe by the European Med- reactions in OoCs may revolutionize in vivo studies by increas- icines Agency (EMA)’s Safety Working Party – and it was con- ing the throughput of analyses, and acknowledged the existence at firmed in the ORCHID workshop. The typical qualification pro- present of a gap between hardware development and the absolute cess applied to OoC devices would foresee a comparison between quantification of biological response required, partly because the OoC data and corresponding data derived from conventional pre- physical, chemical or electrical responses to bioreactions in OoCs clinical drug development models (e.g., animal models, cell sus- 662 ALTEX 36(4), 2019
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